Construction of systematic gene knockout, gene knockdown, and gene overexpression libraries during the past several years has greatly simplified analysis of gene functions both in human cell culture system and model organisms. However, these libraries do not contain interesting loss- and gain-of-function point mutation alleles, which would be invaluable for modeling analysis of numerous existing natural allelic variants and acquired mutations for their contributions to human diseases and response to therapeutic drugs in human pathogens. To facilitate such studies, a long-term goal of this proposed project is to build a "functional variomics" tool in both human and model organisms, with potentially numerous variant alleles being pre-made for each gene in a genome by random mutagenesis and used for isolating alleles that confer phenotypes of interest. However, due to anticipated technical challenges, this tool will be first built and tested in the model organism Saccharomyces cerevisiae to prove the concepts and to provide key parameters. The physical resource, the genome-wide random mutagenesis libraries, will be built mostly by directly cloning error-prone PCR products in yeast cells via homologous recombination. This will be done individually for each of the ~6,600 protein-encoding genes in the yeast genome and the experiments will be carried out in a 96-well high throughput format. Once available, these random mutagenesis libraries will be studied as ONE single population to rapidly and systematically identify all yeast genes whose mutations (either loss- or gain-of-function alleles) cause drug resistance. This will take advantage of microarray analysis of the unique "molecular barcodes" or "tags" to be built into each individual mutagenesis library. Genes identified from such a systematic study will be validated by re- isolating drug resistant clones from the individual mutagenesis libraries. Key amino acid substitutions that contribute to drug resistance will also be identified with sequence-verification of resistant alleles. The validated drug resistant genes will also be used to identify the unknown cellular targets of several drugs because mutations in drug targets represent a major underlying mechanism for drug resistance. Proving the technological concept in yeast will pave the way for developing similar tools in other systems, including both the human cell culture system and human pathogens. The actual mechanisms of drug resistance and drug actions learned from yeast will also provide invaluable hints for rapidly elucidating similar mechanisms in these other systems. PUBLIC HEALTH RELEVANCE: Building a visionary "functional variomics" tool first in the model organism yeast will prove technical concepts and provide key technical parameters for building similar tools in both human cell culture systems and human pathogens, and such tools will be invaluable for modeling analysis of numerous existing natural allelic variants and acquired mutations for their contributions to human diseases and response to therapeutic drugs in human pathogens. The actual mechanisms of drug resistance and drug modes of actions learned from yeast will also provide invaluable hints for rapidly elucidating similar mechanisms in other systems.